Molecular Dissection of Melatonin Synthesis In Vivo

体内褪黑素合成的分子解剖

• 批准号: 6529782
• 负责人: JIMO BORJIGIN
• 金额: $ 27.3万
• 依托单位: CARNEGIE INSTITUTION OF WASHINGTON, D.C.
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6529782
• 关键词: acyltransferase; aromatic L aminoacid decarboxylase; biological signal transduction; cAMP response element binding protein; circadian rhythms; cyclic AMP; enzyme inhibitors; gene expression; genetically modified animals; hormone biosynthesis; hormone regulation /control mechanism; immunoprecipitation; laboratory rat; melatonin; methyltransferase; microdialysis; phosphorylation; pineal body; protein degradation; protein kinase A; protein structure function; serotonin; site directed mutagenesis; transfection; tryptophan 5 monooxygenase; western blottings

DESCRIPTION: (provided by applicant) Melatonin is a nocturnal hormone rhythmically synthesized and released from the pineal gland due to the actions of four enzymes: tryptophan hydroxylase (TPFI), aromatic amino acid decarboxylase (AAADC), serotonin N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT). Although current evidence suggests that in rats cAMP signaling mediates both transcriptional and post-transcriptional control of melatonin formation, little is known about the in vivo targets of cAMP in the pineal. We hypothesize that cAMP regulates melatonin synthesis principally by PKA activation, which phosphorylates key proteins involved in the biosynthesis of melatonin; phosphorylation of these proteins results in a combination of increased transcription and decreased degradation of these biosynthetic enzymes. We plan to investigate the role of the cAMP signaling pathway in pineal circadian rhythms in the intact animal by using an integrated molecular and physiological approach. Aim 1 will establish the importance of the cAMP-dependent protein kinase (PKA) and CREB in transcriptional activation of NAT and melatonin formation in vivo. We will determine the effects of pharmacological inhibitors and activators of PKA catalysis in vivo by microdialysis and examine their influence on NAT transcriptional activation and repression during the day and night. We will also deliver recombinant adenoviral vectors expressing A-CREB or constitutively active PKA into the intact pineals to examine their effects on NAT mRNA and melatonin production. Aim 2 will evaluate the role of FKA in NAT protein stability in vivo. We will utilize a phospho-NAT specific antibody to study the functional significance of PKA-mediated NAT phosphorylation; we will characterize NAT from a mutant strain of rat we discovered that has lower NAT protein level due to a point mutation in a PKA phosphorylation site; lastly, we will use in vivo viral vector delivery and in vivo on-line microdialysis to study the stability and function of NAT mutants. Aim 3 will characterize the role of cAMP in the generation of our newly discovered tri-phasic circadian serotonin release in the pineal in vivo by again utilizing pharmacologic and molecular manipulations of cAMP signaling combined with in vivo physiological measurements of serotonin rhythms. These experiments will further our understanding of the in vivo signal transduction mechanisms that control transcriptional and post-transcriptional activation of melatonin and serotonin formation, which may play a role in the pathogenesis of sleep, psychiatric, and neurological disorders.

描述：（由申请人提供）褪黑激素是一种夜间激素 由于动作而有节奏地合成并从松果体释放 四种酶：色氨酸羟化酶 (TPFI)、芳香氨基酸 脱羧酶 (AAADC)、血清素 N-乙酰转移酶 (NAT) 和 羟基吲哚-O-甲基转移酶（HIOMT）。尽管目前的证据表明 在大鼠中，cAMP 信号介导转录和 褪黑激素形成的转录后控制，目前知之甚少 松果体中 cAMP 的体内靶标。 我们假设 cAMP 主要通过 PKA 调节褪黑激素的合成 激活，磷酸化参与生物合成的关键蛋白质 褪黑激素；这些蛋白质的磷酸化导致组合 这些生物合成物的转录增加并减少降解 酶。我们计划研究 cAMP 信号通路在 通过使用集成分子来研究完整动物的松果体昼夜节律 和生理方法。目标 1 将确定以下内容的重要性： cAMP 依赖性蛋白激酶 (PKA) 和 CREB ​​在转录激活中的作用 NAT 和体内褪黑激素的形成。我们将确定影响 体内 PKA 催化的药理学抑制剂和激活剂 微透析并检查它们对 NAT 转录激活的影响 白天和晚上的镇压。我们还将提供重组 表达 A-CREB ​​或组成型活性 PKA 的腺病毒载体进入 完整的松果体，以检查它们对 NAT mRNA 和褪黑激素产生的影响。 目标 2 将评估 FKA 在体内 NAT 蛋白稳定性中的作用。我们将 利用磷酸 NAT 特异性抗体来研究 PKA 介导的 NAT 磷酸化；我们将表征突变株的 NAT 我们发现，由于点突变，大鼠的 NAT 蛋白水平较低 在 PKA 磷酸化位点；最后，我们将使用体内病毒载体 递送和体内在线微透析以研究稳定性和功能 NAT 突变体。目标 3 将描述 cAMP 在生成 我们新发现的松果体中三相昼夜血清素释放 体内再次利用 cAMP 的药理学和分子操作 信号传导与体内血清素生理测量相结合 节奏。这些实验将进一步我们对体内信号的理解 控制转录和转录后的转导机制 激活褪黑激素和血清素的形成，这可能在 睡眠、精神和神经系统疾病的发病机制。